Previously, in studies involving a semi-homologous system of gnotobiotic newborn pigs and a virulent porcine rotavirus strain (SB-1A) and an avirulent human rotavirus strain (DS-1) and their reassortants, we demonstrated that: (i) the third (VP3), fourth (VP4), ninth (VP7), and tenth (NSP4) porcine rotavirus gene each play an important independent role in the virulence of rotavirus infection in piglets; and (ii) all four of the porcine rotavirus virulence-associated genes are required for the induction of diarrhea and the shedding of rotavirus by piglets. These observations suggested a potential new strategy for attenuation of wild-type human rotaviruses of major epidemiologic importance and its application to the development of a safe and effective vaccine. Previously, we developed rhesus (RRV)-, bovine (UK)-, and porcine (Gottfried)-based multivalent vaccine candidates which were designed to provide antigenic coverage for VP7 (G) serotypes 1-4, 8 and 9 of epidemiologic importance. This year, in order to study which gene(s) of a murine rotavirus EB strain are involved in induction of diarrhea in a homologous mouse model, we passaged a cell culture-adapted avirulent (non-diarrheagenic) murine rotavirus EB strain serially in 4-5 day-old CD-1 mice until the virus became virulent (diarrheagenic). This virulent virus was then inoculated onto primary African green monkey kidney (AGMK) cells and passaged serially 18 times. The 18th cell culture-passaged virus that was shown to be avirulent in mouse pups became virulent again during subsequent serial passage in CD-1 mice. This virulent virus when passaged again serially 18 times in primary AGMK cells became avirulent in mice. Sequencing of all 11 genes of selected virulent and avirulent viruses generated in this study, revealed that virulent viruses when compared with avirulent viruses bore (i) distinct mutations in gene 4 (encoding outer capsid VP4) and in gene 10 (encoding viral enterotoxin NSP4) and (ii) no significant mutations in other genes. In addition, no significant difference in virus replication efficiency in mice was observed between virulent and avirulent viruses. We generated 3 baculovirus recombinants expressing the NSP4 protein derived from high mouse-passaged virulent EB virus (sample A), low mouse-passaged virulent EB virus (sample B), or cell culture-passaged avirulent EB virus (sample C). Upon oral inoculation in mouse pups, the development of diarrhea was observed in 91.6% (11 of 12) in sample A group; 12.5% (1 of 8) in sample B group; and 0% (0 of 12) in sample C group, indicating that the NSP4 protein played an important role in pathogenesis in this model.